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Exposure of lipopolysaccharide triggers macrophage pro-inflammatory polarization accompanied by metabolic reprogramming, characterized by elevated aerobic glycolysis and a broken tricarboxylic acid cycle. However, in contrast to lipopolysaccharide, CD40 signal is able to drive pro-inflammatory and anti-tumorigenic polarization by some yet undefined metabolic programming. Here we show that CD40 activation triggers fatty acid oxidation (FAO) and glutamine metabolism to promote ATP citrate lyase-dependent epigenetic reprogramming of pro-inflammatory genes and anti-tumorigenic phenotypes in macrophages. Mechanistically, glutamine usage reinforces FAO-induced pro-inflammatory and anti-tumorigenic activation by fine-tuning the NAD+/NADH ratio via glutamine-to-lactate conversion. Genetic ablation of important metabolic enzymes involved in CD40-mediated metabolic reprogramming abolishes agonistic anti-CD40-induced antitumor responses and reeducation of tumor-associated macrophages. Together these data show that metabolic reprogramming, which includes FAO and glutamine metabolism, controls the activation of pro-inflammatory and anti-tumorigenic polarization, and highlight a therapeutic potential of metabolic preconditioning of tumor-associated macrophages before agonistic anti-CD40 treatments. ispartof: NATURE IMMUNOLOGY vol:24 issue:3 pages:452-+ ispartof: location:United States status: published

Characterization of 22Rv1-derived sublines for invasion activity and galectin-4 expression level.

Supplementary methods and figure legends

Metastatic prostate cancer continues to pose a difficult therapeutic challenge. Prostate cancer progression is associated with aberrant O-glycosylation of cancer cell surface receptors, but the functional impact of such events is uncertain. Here we report spontaneous metastasis of human prostate cancer xenografts that express high levels of galectin-4 along with genetic signatures of EGFR-HER2 signaling and O-glycosylation. Galectin-4 expression in clinical specimens of prostate cancer correlated with poor patient survival. Galectin-4 binding to multiple receptor tyrosine kinases stimulated their autophosphorylation, activated expression of pERK, pAkt, fibronectin, and Twist1, and lowered expression of E-cadherin, thereby facilitating epithelial–mesenchymal transition, invasion, and metastasis. In vivo investigations established that galectin-4 expression enabled prostate cancer cells to repopulate tumors in orthotopic and heterotopic tissues. Notably, these effects of galectin-4 relied upon O-glycosylation mediated by C1GALT1, a galactosyltransferase implicated in other cancers. Parallel changes in galectin-4 and O-glycosylation triggered aberrant receptor signaling and more aggressive invasive character in prostate cancer cells, which through better survival in the circulation also contributed to the bulk cell progeny of distal tumors. Our findings establish galectin-4 and C1GALT1-mediated glycosylation in a signaling axis that is activated during prostate cancer progression, with implications for therapeutic targeting of advanced metastatic disease. Cancer Res; 76(19); 5756–67. ©2016 AACR.

Immunoediting sculpts immunogenicity and thwarts host anti-tumor responses in tumor cells during tumorigenesis; however, it remains unknown whether metabolic programming of tumor cells can be guided by immunosurveillance. Here, we report that T cell-mediated immunosurveillance in early-stage tumorigenesis instructs c-Myc upregulation and metabolic reprogramming in tumor cells. This previously unexplored tumor-immune interaction is controlled by non-canonical interferon gamma (IFNγ)-STAT3 signaling and supports tumor immune evasion. Our findings uncover that immunoediting instructs deregulated bioenergetic programs in tumor cells to empower them to disarm the T cell-mediated immunosurveillance by imposing metabolic tug-of-war between tumor and infiltrating T cells and forming the suppressive tumor microenvironment.

Metastatic prostate cancer continues to pose a difficult therapeutic challenge. Prostate cancer progression is associated with aberrant O-glycosylation of cancer cell surface receptors, but the functional impact of such events is uncertain. Here we report spontaneous metastasis of human prostate cancer xenografts that express high levels of galectin-4 along with genetic signatures of EGFR-HER2 signaling and O-glycosylation. Galectin-4 expression in clinical specimens of prostate cancer correlated with poor patient survival. Galectin-4 binding to multiple receptor tyrosine kinases stimulated their autophosphorylation, activated expression of pERK, pAkt, fibronectin, and Twist1, and lowered expression of E-cadherin, thereby facilitating epithelial–mesenchymal transition, invasion, and metastasis. In vivo investigations established that galectin-4 expression enabled prostate cancer cells to repopulate tumors in orthotopic and heterotopic tissues. Notably, these effects of galectin-4 relied upon O-glycosylation mediated by C1GALT1, a galactosyltransferase implicated in other cancers. Parallel changes in galectin-4 and O-glycosylation triggered aberrant receptor signaling and more aggressive invasive character in prostate cancer cells, which through better survival in the circulation also contributed to the bulk cell progeny of distal tumors. Our findings establish galectin-4 and C1GALT1-mediated glycosylation in a signaling axis that is activated during prostate cancer progression, with implications for therapeutic targeting of advanced metastatic disease. Cancer Res; 76(19); 5756–67. ©2016 AACR.

Disseminated castration-resistant prostate cancer (CRPC) is a common disease in men that is characterized by limited survival and resistance to androgen-deprivation therapy. The increase in human epidermal growth factor receptor 2 (HER2) signaling contributes to androgen receptor activity in a subset of patients with CRPC; however, enigmatically, HER2-targeted therapies have demonstrated a lack of efficacy in patients with CRPC. Aberrant glycosylation is a hallmark of cancer and involves key processes that support cancer progression. Using transcriptomic analysis of prostate cancer data sets, histopathologic examination of clinical specimens, and in vivo experiments of xenograft models, we reveal in this study a coordinated increase in glycan-binding protein, galectin-4, specific glycosyltransferases of core 1 synthase, glycoprotein- N-acetylgalactosamine 3-β-galactosyltransferase 1 (C1GALT1) and ST3 beta-galactoside α-2,3-sialyltransferase 1 (ST3GAL1), and resulting mucin-type O-glycans during the progression of CRPC. Furthermore, galectin-4 engaged with C1GALT1-dependent O-glycans to promote castration resistance and metastasis by activating receptor tyrosine kinase signaling and cancer cell stemness properties mediated by SRY-box 9 (SOX9). This galectin-glycan interaction up-regulated the MYC-dependent expression of C1GALT1 and ST3GAL1, which altered cellular mucin-type O-glycosylation to allow for galectin-4 binding. In clinical prostate cancer, high-level expression of C1GALT1 and galectin-4 together predict poor overall survival compared with low-level expression of C1GALT1 and galectin-4. In summary, MYC regulates abnormal O-glycosylation, thus priming cells for binding to galectin-4 and downstream signaling, which promotes castration resistance and metastasis.-Tzeng, S.-F., Tsai, C.-H., Chao, T.-K., Chou, Y.-C., Yang, Y.-C., Tsai, M.-H., Cha, T.-L., Hsiao, P.-W. O-Glycosylation-mediated signaling circuit drives metastatic castration-resistant prostate cancer.

Herbal medicine is a popular complementary or alternative treatment for prostate cancer. Wedelia chinensis has at least three active compounds, wedelolactone, luteolin, and apigenin synergistically inhibiting prostate cancer cell growth in vitro. Here, we report a systematic study to develop a standardized and effect-optimized herbal extract, designated as W. chinensis extract (WCE) to facilitate its future scientific validation and clinical use. Ethanolic extract of dried W. chinensis plant was further condensed, acid hydrolyzed, and enriched with preparative chromatography. The chemical compositions of multiple batches of the standardized preparation WCE were quantified by LC/MS/MS, and biological activities were analyzed by in vitro and in vivo assays. Furthermore, the pharmacokinetics of the holistic WCE were compared with the combination of the equivalent principal active compounds through oral administration. The results indicated that quantitative chemical assay and PSA (prostate-specific antigen)-reporter assay together are suitable to measure the quality and efficacy of a standardized Wedelia extract on a xenograft tumor model. The presence of minor concomitant compounds in WCE prolonged the systemic exposure to the active compounds, thus augmented the anti-tumor efficacy of WCE. In conclusion, a combination of LC/MS/MS and PSA reporter assay is suitable to qualify a standardized preparation of WCE. Furthermore, the pharmacokinetics and oral bioavailability of active compounds demonstrate that holistic WCE exerted additional pharmacological synergy beyond the multi-targeted therapeutic effects caused by more than one active compound. WCE merits a higher priority to be studied for use in prostate cancer treatment.

Activation of the NFκB pathway is often associated with advanced cancer and has thus been regarded as a rational therapeutic target. Wedelia chinensis is rich in luteolin, apigenin, and wedelolactone that act synergistically to suppress androgen receptor activity in prostate cancer. Interestingly, our evaluation of a standardized Wedelia chinensis herbal extract (WCE) concluded its efficacy on hormone-refractory prostate cancer through systemic mechanisms. Oral administration of WCE significantly attenuated tumor growth and metastasis in orthotopic PC-3 and DU145 xenografts. Genome-wide transcriptome analysis of these tumors revealed that WCE suppressed the expression of IKKα/β phosphorylation and downstream cytokines/chemokines, e.g., IL6, CXCL1, and CXCL8. Through restraining the cytokines expression, WCE reduced tumor-elicited infiltration of myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs) and endothelial cells into the tumors, therefore inhibiting angiogenesis, tumor growth, and metastasis. In MDSCs, WCE also reduced STAT3 activation, downregulated S100A8 expression and prevented their expansion. Use of WCE in combination with docetaxel significantly suppressed docetaxel-induced NFκB activation, boosted the therapeutic effect and reduced the systemic toxicity caused by docetaxel monotherapy. These data suggest that a standardized preparation of Wedelia chinensis extract improved prostate cancer therapy through immunomodulation and has potential application as an adjuvant agent for castration-resistant prostate cancer.

Crosstalk between the androgen receptor (AR) and other signaling pathways in prostate cancer (PCa) severely affects the therapeutic outcome of hormonal therapy. Although anti-androgen therapy prolongs overall survival in PCa patients, resistance rapidly develops and is often associated with increased AR expression and upregulation of the HER2/3-AKT signaling pathway. However, single agent therapy targeting AR, HER2/3 or AKT usually fails due to the reciprocal feedback loop. Previously, we reported that wedelolactone, apigenin, and luteolin are the active compounds in Wedelia chinensis herbal extract, and act synergistically to inhibit the AR activity in PCa. Here, we further demonstrated that an herbal extract of W. chinensis (WCE) effectively disrupted the AR, HER2/3, and AKT signaling networks and therefore enhanced the therapeutic efficacy of androgen ablation in PCa. Furthermore, WCE remained effective in suppressing AR and HER2/3 signaling in an in vivo adapted castration-resistant PCa (CRPC) LNCaP cell model that was insensitive to androgen withdrawal and second-line antiandrogen, enzalutamide. This study provides preclinical evidence that the use of a defined, single plant-derived extract can augment the therapeutic efficacy of castration with significantly prolonged progression-free survival. These data also establish a solid basis for using WCE as a candidate agent in clinical studies.

Introduction Thomsen-Friedenreich (TF or T)-related antigens are short O-glycans highly expressed in over 90% of all human cancers including prostate, breast, ovary, bladder, colon, liver and gastric cancers. Especially in prostatic cancer (PCa), a higher level of T antigen was found correlated with the histological grade and bone metastasis; however, its functional impact in PCa and the underlying mechanism remain perplexing. We showed that spontaneous metastasis of human PCa xenografts expresses high levels of galectin-4, along with gene signatures for ERRB receptors signalling and sialylated core 1 O-glycosylation. Galectin-4 expression in clinical PCa is positively correlated with tumour grade, PSA recurrent rate, and poor survival. Galectin-4 binding to multiple receptor tyrosine kinases (RTKs) in PCa cells stimulates their autophosphorylation and downstream signalling pathways, therefore promoting epithelial-mesenchymal transition and invasive tumour growth. Material and methods Orthotopic xenografts of LNCaP PCa cell line were implanted in athymic nude mice to derive a series of castration-resistant prostate cancer (CRPC) cell populations. Gene expression was detected using qRT-PCR, western blotting, and IHC. Manipulation of gene expression was conducted by overexpression and shRNA knockdown approaches with lentivectors in relevance cell populations. Permission for all animal works has been obtained from the Institutional Animal Care and Use Committee, Academia Sinica. Results and discussions Galectin-4 was upregulated during the development of castration resistance and metastasis. High expression of galectin-4 in PCa cells exhibited castration resistance, tumor-regenerating stem-like cells, expressed SOX9 and ALDH1A1, and ability to colonise distant metastases. We identify a feed-forward signalling pathway by which galectin-4 signalling through RTK-Myc axis upregulated gene transcription of enzymes in a specific O-glycosylation pathway and the biosynthesis of their substrates, thus altered the cellular modification of O-glycosylation, and bolstered castration resistance and metastasis. These coordinated effects conferred more and more binding sites for galectin-4 and amplified the RTKs signalling, implicating crosstalk mechanisms to activate androgen receptor signalling and drive the PCa progression. Conclusion MYC regulates oncofetal O-glycosylation in RTKs thus primes the cells for binding to galectin-4 and downstream signalling, which promotes PCa castration resistance, metastasis, and clinically poor survival.

Oncofetal Galβ1-3GalNAc O-glycan, T antigen, is increased in about 60% carcinomas of breast, colon, stomach, and prostate. Notably, coexpression of T antigen with CD44 or CD133, markers of cancer stem cells, has been identified in lung, breast, and liver cancers. Using spontaneous metastasis model of 22Rv1 cells, we derived high metastatic 22Rv1-M4 cells and found altered O-glycosylation in the 22Rv1-M4 cells and also 22Rv1-M4-derived host microenvironment compared to parental cells. Our study indicated that high expressions of T antigen synthase C1GALT1 are associated with metastatic PCa and poor survival, probably contributed by the interaction of galectin-4 and glycan. Galectin-4 engaged with MYC-dependent C1GALT1 mediated O-glycan and resulted in RTK phosphorylation; activation of RTKs enhanced not only androgen-independent growth but also metastatic colonization which mediated by ERK-AP1-mediated SOX9 expression. Downregulation of SOX9 significantly inhibited the tumorsphere formation and in vivo metastasis. Moreover, coexpression of SOX9 and MYC in immortalized prostate epithelial cells RWPE1 generates the heterogeneous population presenting both epithelial type and cancer-like cells in mouse renal capsule. On the other hand, analyses of lung tissues from mice bearing tumor of 22Rv1-M4 cells relative to that bearing tumor of 22Rv1 parental cells exhibit the upregulated O-glycosylation enzymes, which associated with increased plant Jacalin lectin staining, CD45+ cells infiltration, and endothelial permeability indicated by dextran leakage. Tail-vein injection of 22Rv1-M4 cells exhibits aggressive systemic metastasis in a mouse conditioned by subcutaneous 22Rv1-M4 tumor compared to that in a mouse conditioned by subcutaneous 22Rv1 tumor These data suggest high metastatic prostate cancer cells adapt the lung microenvironment for seeding and colonization of cancer cells. Citation Format: Chin-Hsien Tsai, Sheue-Fen Tzeng. Abnormal O-glycosylation promotes prostate cancer stemness and mediates metastatic niche [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr LB-358.

Dysregulation of cell surface proteolysis has been strongly implicated in tumorigenicity and metastasis. In this study, we delineated the role of hepatocyte growth factor activator inhibitor-2 (HAI-2) in prostate cancer (PCa) cell migration, invasion, tumorigenicity and metastasis using a human PCa progression model (103E, N1, and N2 cells) and xenograft models. N1 and N2 cells were established through serial intraprostatic propagation of 103E human PCa cells and isolation of the metastatic cells from nearby lymph nodes. The invasion capability of these cells was revealed to gradually increase throughout the serial isolations (103E

Curcumin has been shown to possess potent chemopreventive and antitumor effects on prostate cancer. However, the molecular mechanism involved in curcumin's ability to suppress prostate cancer cell invasion, tumor growth, and metastasis is not yet well understood. In this study, we have shown that curcumin can suppress epidermal growth factor (EGF)- stimulated and heregulin-stimulated PC-3 cell invasion, as well as androgen-induced LNCaP cell invasion. Curcumin treatment significantly resulted in reduced matrix metalloproteinase 9 activity and downregulation of cellular matriptase, a membrane-anchored serine protease with oncogenic roles in tumor formation and invasion. Our data further show that curcumin is able to inhibit the induction effects of androgens and EGF on matriptase activation, as well as to reduce the activated levels of matriptase after its overexpression, thus suggesting that curcumin may interrupt diverse signal pathways to block the protease. Furthermore, the reduction of activated matriptase in cells by curcumin was also partly due to curcumin's effect on promoting the shedding of matriptase into an extracellular environment, but not via altering matriptase gene expression. In addition, curcumin significantly suppressed the invasive ability of prostate cancer cells induced by matriptase overexpression. In xenograft model, curcumin not only inhibits prostate cancer tumor growth and metastasis but also downregulates matriptase activity in vivo. Overall, the data indicate that curcumin exhibits a suppressive effect on prostate cancer cell invasion, tumor growth, and metastasis, at least in part via downregulating matriptase function. Cancer Prev Res; 6(5); 495–505. ©2013 AACR.

We previously demonstrated that the principal active compounds (wedelolectone, luteolin, and apigenin) in the ethanol extract of Wedelia chinensis synergistically inhibit human prostate carcinoma 22Rv1 cell growth and that this extract therefore has potential for use as a prostate cancer (PCa) therapy. Here, we developed a standardized preparation for a W. chinensis ethanol extract that is enriched in the principal active compounds. A comparison of the effect of herbal treatment, androgen ablation therapy, and a combination of the two on the development of androgen-dependent LNCaP tumors in a mouse model showed that the herbal extract alone decreased androgen-induced tumor growth and when in combination with androgen ablation therapy, resulted in more potent inhibition of both tumor growth and cancer metastasis. Our study demonstrated this herbal remedy simultaneously inhibited androgen receptor, NFκB and HER2/3-AKT signaling pathways to induce cell apoptosis. Importantly, our herbal remedy diminished androgen receptor overexpression and AKT activation induced by androgen ablation thus preventing castration-resistant PCa development. Moreover, the herbal remedy standing-alone curbed chemokine expression from hormone-refractory PC-3 tumor and consequently restrained the mobilization of CD11b+Gr1+ myeloid cells in tumor-bearing mice, which effectively inhibited PCa growth and metastasis. In conclusion, this standardized preparation of W. chinensis ethanol extract improved the outcome of PCa therapy either as an add-on to hormonal therapy for androgen-dependent disease or as a monotherapy for hormone-refractory disease. Citation Format: Chin-Hsien Tsai, Sheue-Fen Tzeng, Pei-Wen Hsiao. Characterized herbal extract of Wedelia chinensis suppresses prostate cancer growth and metastasis by targeting multiple intrinsic pathways and modulating immune cells. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A58. doi:10.1158/1538-7445.CHTME14-A58

Hormone-refractory and metastatic prostate cancer (PCa) both result in therapeutic failure. Developing PCa animal model is critical to understand the cancer progression and to access therapeutic effects. Here, we established the orthotopic xenograft of androgen-independent PCa cell line, 22Rv1, in nude mouse model and cultured the metastatic cancer cells. The same procedure was repeated to evolve highly metastatic cells, 22Rv1-LN3. In experimental metastasis, 22Rv1-LN3 can colonize and grow in the LN, lung and brain through tail vein injection but 22Rv1 cell failed to colonize in these organs. Besides, intracardiac injection of 22Rv1-LN3 also caused bone metastasis. In the model, we found 22Rv1-LN3 cell have higher protease activity (e.g. matriptase and MMP9) along with lower level of protease inhibitors, (e.g. HAI-2, a matriptase inhibitor and TIMP2/TIMP3). Transcriptional level of these genes determined the in vitro invasion activity and in vivo tumorigenicity. Given the differential capacity to seed different tissues, we further analyze the stem cell property by tumorsphere assay and in vivo transplantation with a reduced cell number. Sphere-formation rate in 22Rv1-LN3 cells was 8% compared to only 0.1% in 22Rv1 cells. Tumorigenicity of 22Rv1-LN3 cells in orthotopic transplantation was 100% (8/8) compared to 20% (2/10) in 22Rv1 cells. Moreover, primary tumor of 22Rv1-LN3 cells developed spontaneous metastases in lymph nodes and lung at 8-week, whereas 22Rv1 cells hardly metastasized even at 14-week. As analyzed by qRT-PCR, primary 22Rv1-LN3 tumor vs. 22Rv1-Luc2 induced higher levels of Arginase and iNOS expression in lung tissue, which indicates the proclivity for metastatic niche formation. In conclusion, this PCa progression model not only elevated the malignant properties of cancer cells but also influenced the tumor microenvironment. Citation Format: Sheue-Fen Tzeng, Chin-Hsien Tsai, Ming-Shyue Lee, Pei-Wen Hsiao. High malignant prostate cancer cells developed enhanced tumor-initiating/metastatasis-initiating activity and altered the lung microenvironment. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B85. doi:10.1158/1538-7445.CHTME14-B85